CN113801206A - Method for inducing anti-neocoronavirus neutralizing antibody by using receptor recognition domain - Google Patents

Abstract

The present disclosure relates to methods of inducing neutralizing antibodies against neocoronaviruses using receptor recognition domains. Specifically, the present application provides an immunogenic peptide against a novel coronavirus SARS-CoV-2, which comprises an RBD region of SARS-CoV-2 virus spike protein S or a cysteine-modified RBD region; it encodes nucleotide molecule, carrier and host cell containing the nucleotide molecule. The application also provides vaccines against the novel coronavirus SARS-CoV-2, their preparation and use. The vaccine disclosed by the invention is safe, can continuously generate high-titer neutralizing antibodies, and can be used for preventing and/or treating novel coronavirus infection and symptoms thereof.

Description

Method for inducing anti-neocoronavirus neutralizing antibody by using receptor recognition domain

Technical Field

The present disclosure is in the field of biotechnology and vaccines. In particular, the disclosure relates to methods of inducing neutralizing antibodies against the novel coronavirus (SARS-CoV-2) using a receptor recognition domain.

Background

To date, three highly pathogenic human coronaviruses (CoVs) have been identified, including the middle east respiratory syndrome coronavirus (MERS-CoV), the Severe Acute Respiratory Syndrome (SARS) coronavirus (SARS-CoV), and a 2019 novel coronavirus (SARS-CoV-2, new coronavirus for short).

The interpersonal transmission rate of the new coronavirus has exceeded SARS-CoV and MERS-CoV. WHO announced that a global pandemic has occurred in new crown blight at 11/3/2020. At present, the intermediate host of the new coronaviruses is still unknown, and there is no effective preventive or therapeutic means. Therefore, the development of vaccines and drugs for the prevention and treatment of new coronaviruses is a key issue that needs to be addressed urgently.

It is well known that vaccines have been the most effective and economical measure in the prevention of infectious diseases since their advent, and most of the currently used preventive vaccines are aimed at activating neutralizing antibodies. Therefore, in order to develop a vaccine against the new coronavirus, an immunogen capable of effectively activating neutralizing antibodies needs to be found.

Coronaviruses comprise four structural proteins, including the spike protein (S protein), the envelope protein, the membrane protein, and the nucleocapsid protein. Among them, the S protein plays the most important role in the attachment, fusion and entry processes of viruses, and is also a main target of antibodies, entry inhibitors and vaccines. The S protein mediates entry of the virus into the host cell by first binding to the host receptor via the Receptor Binding Domain (RBD) of the S1 subunit, and then fusing the virus and host cell membranes via the S2 subunit.

WHO's COVID-19 global research roadmap states: animals immunized with coronavirus vaccines may develop more severe symptoms when exposed to live virus again. Non-neutralizing antibodies or lower antibody levels produced by vaccine immunization may cause antibody-dependent enhancement (ADE) effects that enhance viral pathogenicity. In summary, neutralizing antibodies block binding of the virus to cell surface receptors, neutralizing the ability of the virus to infect; non-neutralizing antibodies bind to the virus but in some cases, the antibody Fc fragment binds to a cell surface Fc receptor, allowing it to infect Fc receptor expressing cells. Therefore, to reduce the side effects of ADE, the RBD region in the S protein of the novel coronavirus would be the most effective target for vaccine development.

The native conformation of RBD in S protein is a trimer, and if RBD is purified alone, it is less supported by the trimer of the S2 subunit, and its conformation is likely to be altered. How to maintain the conformation of the RBD is therefore a problem to be solved.

There is an urgent need in the art to develop novel vaccines that can efficiently generate neutralizing antibodies to novel coronaviruses.

Disclosure of Invention

The present disclosure uses the RBD specific region of the new coronavirus as an immunogen, and simultaneously performs a series of modifications on the RBD region, such as adding disulfide bonds, fusion proteins, and fusion cytokines, thereby making it possible to effectively induce neutralizing antibodies against SARS-CoV-2.

In one aspect of the disclosure, an immunogenic peptide against a novel coronavirus SARS-CoV-2 is provided, comprising an RBD region of the spike protein S of SARS-CoV-2 virus, which RBD region is further modified with cysteine to form an sRBD region.

In some embodiments, the cysteine modification is the addition of a pair of cysteines at the root of the RBD domain to enable disulfide bond formation.

In some embodiments, the immunogenic peptide is selected from the group consisting of: (a) has the sequence shown in SEQ ID NO: 2. 4, 8 or 10; (b) a polypeptide homologous to the polypeptide of (a), e.g., having greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, greater than or equal to 99% homology to SEQ ID No. 2, 4, 8 or 10; (c) and (b) the protein or polypeptide which is derived from the (a) and has immunogenicity through substituting, deleting or adding one or more amino acids in the amino acid sequence defined by the (a).

In some embodiments, the amino acid sequence of the RBD region is set forth in SEQ ID No. 2 and the amino acid sequence of the cysteine-modified sRBD region is set forth in SEQ ID No. 4.

In some embodiments, the RBD region or cysteine-modified sRBD region is comprised in a fusion peptide, e.g., the portion fused thereto includes: proteins of viral or host origin, transferrin (Fn), HIV p24, the stem of enveloped viruses, such as influenza HA2, HIV gp41, antibody Fc-fragments, GM-CSF, IL-21, CD40L or CD40 antibodies.

In some embodiments, the fusion peptide may further comprise: signal peptide, linker peptide, tag, etc. For example, the signal peptide may be selected from: protein itself, CD33, CD8, CD16, mouse IgG1 antibody, influenza HA. The linker peptide may be selected from: (G4S)₃、(G4S)_nGSAGSAAGSGEF, (Gly)6, EFPKPSTPPGSSGGAP, KESGSVSSEQLAQFRSLD, (Gly)8, EGKSSGSGSESKST. The label may be selected from: his-tag, AviTag, Calmodulin tag, polyglutamate tag, E-tag, FLAG tag, HA-tag, Myc-tag, S-tag, SBP-tag, Sof-tag 1, Sof-tag3, Strep-tag, TC tag, V5 tag, T7 tag, VSV tag, Xpress tag, 3X FLAG tag, Isopep tag, Spytag, Snoop tag, and PNE tag.

In some embodiments, the immunogenic peptide is sRBD-hFn, sRBD-HA2, or the like.

In some embodiments, the fusion peptide has the sequence shown in SEQ ID NO 8 or SEQ ID NO 10.

In one aspect of the disclosure, a nucleotide molecule is provided that encodes an immunogenic peptide of the disclosure.

In some embodiments, the nucleotide molecule is selected from the group consisting of: (i) the sequence is shown as SEQ ID NO: 1. 3, 7 or 9; (ii) (ii) a molecule that hybridizes to (i) under stringent conditions; (iii) (iii) a nucleotide molecule having greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, greater than or equal to 99% homology with the sequence in (i) or (ii); (iv) a nucleotide molecule which is formed by substituting, deleting or adding one or more nucleotides in the nucleotide sequence defined by (i) or (ii) and can express a functional RBD immunogenic peptide.

In some embodiments, the coding sequence for the RBD region is shown in SEQ ID NO. 1, the coding sequence for the cysteine modified RBD region is shown in SEQ ID NO. 3, or the sequence of the nucleotide molecule is shown in SEQ ID NO. 7 or SEQ ID NO. 9.

In some embodiments, the nucleotide molecule further comprises a sequence encoding a moiety fused to the RBD region or to the cysteine-modified RBD region.

In one aspect of the disclosure, a vector is provided comprising a nucleotide molecule of the disclosure.

In some embodiments, the carrier is: viral vectors, such as poxviruses (e.g., Temple strains, North American vaccine strains, Whitmania derived strains, Listeria strains, Ankara derived strains, Copenhagen strains, and New York strain poxviruses), adenoviruses (e.g., Ad5, Ad11, Ad26, Ad35, Ad68), lentiviral vectors, adeno-associated viruses, herpes simplex viruses, measles viruses, reoviruses, rhabdoviruses, forest encephalitis viruses, influenza viruses, respiratory syncytial viruses, poliovirus vectors.

In one aspect of the disclosure, a host cell is provided comprising a vector of the disclosure and/or capable of expressing an immunogenic peptide of the disclosure.

In some embodiments, the host cell is a mammalian cell or an insect cell, such as HEK293, HeLa, K562, CHO, NS0, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, and MRC-5 cells; high Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-672302, Hz2E5 and Ao 38.

In some embodiments, the host cell is K562. In some embodiments, the K562 cells comprise sRBD-HA 2.

Thus, also provided in the present disclosure is a cell displaying on its cell membrane surface the novel coronavirus SARS-CoV-2 immunogenic peptide described herein. In some embodiments, the cell comprises a vector having an immunogenic peptide coding sequence described herein. In some embodiments, the cell is transformed with a vector having an immunogenic peptide coding sequence. In some embodiments, the cell has an intact membrane structure displaying the immunogenic peptide. In some embodiments, the cell is an inactivated cell, e.g., using physical inactivation such as X-ray radiation, ultraviolet radiation; or chemical inactivation such as beta-propiolactone, formaldehyde, paraformaldehyde fixation.

In one aspect of the present disclosure, a vaccine against the novel coronavirus SARS-CoV-2 is provided, comprising an immunogenic peptide, nucleotide molecule, vector and/or host cell of the present disclosure.

In one aspect of the disclosure, there is also provided the use of the immunogenic peptides, nucleotide molecules, vectors and/or host cells of the disclosure in the preparation of a vaccine for the prevention or treatment of the novel coronavirus SARS-CoV-2.

In one aspect of the disclosure, there is also provided an immunogenic peptide, nucleotide molecule, vector and/or host cell of the disclosure for use in the prevention or treatment of the novel coronavirus SARS-CoV-2.

In one aspect of the present disclosure, there is also provided a method of preventing or treating a novel coronavirus SARS-CoV-2, the method comprising administering to a subject in need thereof an immunogenic peptide, nucleotide molecule, vector, host cell and/or vaccine of the present disclosure.

In some embodiments, the vaccine is a nucleic acid vaccine (DNA or RNA vaccine), a recombinant protein subunit vaccine, a recombinant viral vector vaccine, a recombinant bacterial vector vaccine, a virus-like particle vaccine, a nanoparticle vaccine, a cell vector vaccine.

In some embodiments, the vaccine comprises or is combined with an adjuvant including, but not limited to: aluminum adjuvant, cholera toxin and its subunit, oligodeoxynucleotide, manganese ion adjuvant, colloidal manganese adjuvant, Freund's adjuvant, SAS adjuvant, MF59 adjuvant, QS-21 adjuvant, Poly I: C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21, etc.

In some embodiments, the vaccine is in a form suitable for vaccination as follows: intramuscular inoculation, intradermal inoculation, subcutaneous inoculation, nasal drops, aerosol inhalation, reproductive tract, rectal, oral, or any combination thereof.

In some embodiments, vaccination is with one or more of the vaccines, e.g., combined vaccination or sequential vaccination.

In some embodiments, one or more of the vaccines are used to vaccinate against other novel coronaviruses, e.g., the other vaccines include vaccines against coronavirus S or S1, e.g., S or S1 is from a source including, but not limited to, SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV, and the like.

In some embodiments, the vaccine comprises a nucleic acid vaccine (DNA or RNA vaccine) in combination with a recombinant human-derived cell vector vaccine.

In some embodiments, the vaccine comprises pcDNA3.1-sRGB-hFn in combination with K562-HA 2-sRGB. In some embodiments, the components of the vaccine combination are administered sequentially, preferably first, before and after administration of the DNA vaccine.

In one aspect of the present disclosure, there is provided a method of preparing a vaccine against a novel coronavirus SARS-CoV-2, the method comprising:

(a) providing an immunogenic peptide, nucleotide molecule, vector and/or host cell of the present disclosure;

(b) combining the active substance provided in (a) with an immunologically or pharmaceutically acceptable carrier.

Any combination of the foregoing aspects and features may be made by those skilled in the art without departing from the spirit and scope of the present disclosure. Other aspects of the disclosure will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

The present disclosure is further described with reference to the accompanying drawings, wherein the showings are for the purpose of illustrating embodiments of the disclosure only and not for the purpose of limiting the scope of the disclosure.

FIG. 1: construction of S protein and sRBD-hFn (disulfide bond modification) eukaryotic expression vector and expression of protein:

FIG. 1A is the construction map of pcDNA3.1-sRGB-hFn and pcDNA3.1-S eukaryotic expression vector plasmid;

FIG. 1B shows that pcDNA3.1-sRBD-hFn and pcDNA3.1-S proteins were successfully expressed in 293T cells.

FIG. 2 construction of lentiviral expression vectors pHAGE-S-puro and pHAGE-sRBD-HA2-puro and validation of expression of S protein and sRBD-HA2 displayed on K562 cell membrane:

FIG. 2A. plasmid construction profiles of pHAGE-S-puro and pHAGE-sRBD-HA2-puro lentivirus expression vectors;

FIG. 2B shows the successful expression of pHAGE-S-puro and pHAGE-sRBD-HA2-puro proteins;

srbd is capable of binding to the ACE2 receptor.

FIG. 3: induction effect of RBD protein on mouse binding and neutralizing antibodies:

the mice used in the experiment are 6-8 weeks old C57/BL6, the immunogen is new coronavirus RBD protein and pcDNA3.1-S plasmid, and the protein is strengthened by adopting aluminum adjuvant.

Figure 3a. elisa method detects the titer of bound antibody in mouse sera at week 2 after the end of immunization. The abscissa is the immunization group and the ordinate is the titer of the bound antibody;

FIG. 3B.293T-ACE2 cells were tested for neutralizing antibody titers in mouse serum at week 4 after immunization. The abscissa is the immune group and the ordinate is the titer (ID) of neutralizing antibodies₅₀). Denotes p<0.05。

FIG. 4: the induction effect of plasmid pcDNA3.1-sRGB-hFn on mouse binding and neutralizing antibodies:

the mouse used in the experiment is C57/BL6 with the age of 6-8 weeks, and the immunogen is the new coronavirus S plasmid pcDNA3.1-S and the modified RBD plasmid pcDNA3.1-sRGB-hFn.

Figure 4a. elisa method to detect the titer of bound antibody in mouse sera at week 2 after the end of immunization. The abscissa is the immunization group and the ordinate is the titer of the bound antibody;

FIG. 4B.293T-ACE2 cells measure the titer of neutralizing antibodies in mouse serum at week 2 after the end of immunization. The abscissa is the immune group and the ordinate is the titer (ID) of neutralizing antibodies₅₀)。

FIG. 5: the induction effect of K562-HA2-sRBD and K562-S cells on mouse binding and neutralizing antibodies:

the mice used in the experiment are C57/BL6 with the age of 6-8 weeks, and the immunogens are K562-HA 2-sRGB and K562-S.

Figure 5a. elisa method to detect the titer of bound antibody in mouse sera at week 2 after the end of immunization. The abscissa is the immunization group and the ordinate is the titer of the bound antibody;

FIG. 5B.293T-ACE2 cells measure the titer of neutralizing antibodies in mouse serum at week 2 after the end of immunization. The abscissa is the immune group and the ordinate is the titer (ID) of neutralizing antibodies₅₀)。

FIG. 6 induction effect of sRBD-hFn protein on mouse-bound and neutralizing antibodies:

the mice used in the experiment are 6-8 weeks old C57/BL6, and the immunogen is sRGB-hFn protein.

Figure 6a. elisa method to detect the titer of bound antibody in mouse sera at week 1 after immunization. The abscissa is the immunization group and the ordinate is the titer of the bound antibody;

FIG. 6B.293T-ACE2 cells were tested for neutralizing antibody titers in mouse serum at week 1 after immunization. The abscissa is the immune group and the ordinate is the titer (ID) of neutralizing antibodies₅₀)。

Detailed Description

The disclosure relates to the field of vaccines, in particular to a method for inducing anti-new coronavirus (SARS-Cov-2) neutralizing antibodies by using RBD regions. The use of the RBD region of the virus, engineered RBD region, RBD region fused to other proteins or cytokines as an immunogen reduces the antibody-dependent enhancement effect (ADE) caused by non-neutralizing antibodies or lower antibody levels, thereby preventing infection by new corona viruses. Animal experiment results prove that the vaccine disclosed by the invention is safe, can continuously generate high-titer neutralizing antibodies, and can be used for preventing and treating the new coronavirus.

All numerical ranges provided herein are intended to expressly include all numbers between the end points of the ranges and numerical ranges there between. The features mentioned in the present disclosure or the features mentioned in the embodiments can be combined. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

As used herein, "about" in the context of a value or range means ± 10% of the recited or claimed value or range.

It is to be understood that when ranges of parameters are provided, the invention likewise provides all integers and decimals thereof within the ranges. For example, "0.1-2.5 mg/day" includes 0.1 mg/day, 0.2 mg/day, 0.3 mg/day, etc. up to 2.5 mg/day.

As used herein, "comprising," having, "or" including "includes" comprising, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …", and "consisting of … …" are subordinate concepts of "comprising", "having", or "including".

RBD immunogenic peptides and molecules encoding same

As used herein, the terms "RBD immunogenic peptide", "immunogenic peptide against the novel coronavirus SARS-CoV-2", and "immunogenic peptide of the present disclosure/application" and the like, which are used interchangeably, refer to a peptide that includes the RBD region or cysteine-modified RBD region (also referred to herein as sRBD region) of the spike protein S of SARS-CoV-2 virus and has the effect of eliciting binding and neutralizing antibodies.

In some embodiments of the disclosure, the immunogenic peptide may be: (a) has the sequence shown in SEQ ID NO: 2. 4, 8 or 10; (b) a polypeptide homologous to the polypeptide of (a), e.g., having greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, greater than or equal to 99% homology to SEQ ID No. 2, 4, 8 or 10; (c) and (b) the protein or polypeptide which is derived from the (a) and has immunogenicity through substituting, deleting or adding one or more amino acids in the amino acid sequence defined by the (a).

In some cases, the immunogenic peptide can include additional moieties linked to the RBD region or the cysteine-modified RBD region, for example, to enhance the stability of the RBD region, enhance neutralizing antibody responses, form multimers, increase cellular responses, and the like. Moieties to which the RBD region may be attached, modified or unmodified, include, but are not limited to: proteins derived from viruses or hosts, transferrin (Fn), HIV p24, and the stem of enveloped viruses, such as influenza HA2, HIV gp41, antibody Fc fragment, GM-CSF, IL-21, CD40L, or CD40 antibody. For example, the sequence of the fusion peptide is shown as SEQ ID NO. 8 or SEQ ID NO. 10.

In some cases, elements such as signal peptides, linkers, molecular tags, and the like may also be included in the fusion peptide. For example, a signal peptide element can refer to an amino acid sequence having the function of directing secretion, localization and/or delivery of a fusion protein, which is typically 5-30 amino acids in length. In some embodiments, the signal peptide element may be selected from: a protein self signal peptide, a CD33 protein signal peptide, a CD8 protein signal peptide, a CD16 protein signal peptide, a mouse IgG1 antibody signal peptide and an influenza HA protein signal peptide. For example, a linker peptide sequence (or linker) can be a short peptide that functions to link different elements in the fusion protein herein, and is typically 1-50 (e.g., 5-50, 5-40, 10-40) amino acids in length. In general, the linker peptide does not affect or seriously affects the formation of the correct fold and spatial conformation of the fusion protein of the invention. In some embodiments, the linker peptide element may be selected from: (G)₄S)₃Joint (G4S)_n、GSAGSAAGSGEF、(Gly)6、EFPKPSTPPGSSGGAP、 KESGSVSSEQLAQFRSLD、(Gly)8、EGKSSGSGSESKST。

The immunogenic peptide may also include variants thereof, such as deletion, insertion and/or substitution of one or more (typically 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids, and addition of one or several (typically up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein or polypeptide. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein or polypeptide.

Immunogenic peptides can be produced by recombinant expression under appropriate circumstances and conditions, e.g., produced by the coding nucleotide molecules, vectors, host cells of the disclosure; it can also be obtained by chemical synthesis or the like, so long as it has the desired amino acid sequence and immunogenicity and reactivity.

As used herein, the terms "immunogenic peptide coding molecule", "RBD coding sequence", and the like, are used interchangeably and all refer to a nucleotide molecule that encodes an immunogenic peptide described in the present disclosure. The nucleic acid molecule may be selected from, for example: (i) the sequence is shown as SEQ ID NO: 1. 3, 7 or 9; (ii) (ii) a molecule that hybridizes to (i) under stringent conditions; (iii) (iii) a nucleotide molecule having greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, greater than or equal to 99% homology with the sequence in (i) or (ii); (iv) a nucleotide molecule which is formed by substituting, deleting or adding one or more nucleotides in the nucleotide sequence defined by (i) or (ii) and can express a functional RBD immunogenic peptide.

As used herein, the term "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 50%, preferably 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, more preferably 95% or more.

The full-length nucleotide sequence or a fragment thereof of the present disclosure can be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in the present disclosure, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Vectors and host cells

The disclosure also relates to vectors comprising the nucleotide molecules encoding RBD, and host cells genetically engineered with the vectors.

The coding sequences of the present disclosure can be used to express or produce recombinant immunogenic peptides by conventional recombinant DNA techniques (Science, 1984; 224: 1431). Generally, the following steps are performed:

(1) transferring the coding nucleotide molecules of the present disclosure, or the recombinant expression vectors containing the nucleotide molecules, into suitable host cells;

(2) a host cell cultured in a suitable medium;

(3) isolating and purifying the protein or polypeptide from the culture medium or the cells.

In the present disclosure, the terms "vector" and "recombinant expression vector" are used interchangeably and refer to a bacterial plasmid, phage, yeast plasmid, animal cell virus, mammalian cell virus or other vector well known in the art. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Expression vectors containing the RBD immunogenic peptide coding sequence and appropriate transcriptional/translational control signals can be constructed using methods conventional in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Expression systems such as pcDNA3.1 vector, pIRES2-EGFP vector, AdMaxTM, and the like may be employed in the present disclosure.

In addition, the expression vector may contain one or more selectable marker genes to provide phenotypic traits useful for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform an appropriate host cell so that it can express the protein or polypeptide. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as animal cells. Representative examples are: escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; animal cells, and the like. In the present disclosure, for example, a host cell selected from the group consisting of: HEK293, HeLa, CHO, K562, NS0, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19 and MRC-5 cells; high Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-672302, Hz2E5 and Ao 38.

The nucleotide molecules of the present disclosure will provide enhanced transcription when expressed in higher eukaryotic cells if enhancer sequences are inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene. It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

The recombinant polypeptide in the above method may be expressed or secreted intracellularly or on the cell membrane to the outside of the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Vaccines and immunoconjugates

Also provided herein is a vaccine, or immunogenic composition, comprising the immunogenic peptides, nucleotide molecules, vectors and/or host cells of the disclosure. The vaccine comprises a formulation of the immunogenic peptides and/or nucleic acid molecules of the present disclosure in a form capable of being administered to a vertebrate, preferably a mammal, and which induces a protective immune response that enhances immunity to prevent and/or alleviate the novel coronavirus and/or at least one symptom thereof.

The term "protective immune response" or "protective response" refers to an immune response to an infectious agent or disease that is exhibited by a vertebrate (e.g., a human), which prevents or reduces infection or reduces at least one disease symptom thereof, mediated by an immunogen.

The term "vertebrate" or "subject" or "patient" refers to any member of the subphylum chordata, including, but not limited to: humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; livestock such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds include domesticated, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese. The terms "mammal" and "animal" are included in this definition and are intended to encompass adult, juvenile, and newborn individuals.

The vaccines herein can be recombinant protein vaccines, recombinant DNA vaccines, recombinant viral vector vaccines (e.g., adenoviral vectors, poxvirus vectors, adeno-associated viral vectors, herpes simplex viral vectors, cytomegalovirus vectors), recombinant bacterial vector vaccines, recombinant yeast vector vaccines, or recombinant virus-like particle vaccines. In some embodiments, the vaccine herein is selected from a recombinant DNA vaccine, a recombinant adenoviral vector, a recombinant poxvirus vector, or a combination of one or two or three thereof.

In some embodiments, one or more vaccines or combinations thereof selected from the group consisting of: recombinant plasmid vaccines (DNAs), such as DNA vaccines comprising cysteine-modified RBD region coding sequences fused to human recombinant Ferritin or HA2 (e.g., pcdna3.1-sRBD-hFn); recombinant protein subunit vaccines (proteins), such as RBD proteins (not comprising disulfide bond modifications), cysteine-modified RBD proteins fused to human recombinant Ferritin or HA2 (sRBD-hFn protein, sRBD-HA2 protein); recombinant human cell vector vaccines, such as K562-HA 2-sRGB.

An effective amount of the immunogen herein is included in the vaccine compositions herein. The vaccine compositions of the present disclosure comprise an immunogen in an amount sufficient to achieve the desired biological effect. The term "effective amount" generally refers to an amount of immunogen that can induce a protective immune response sufficient to induce immunity to prevent and/or alleviate an infection or disease and/or to reduce at least one symptom of an infection or disease.

Adjuvants may also be included in the vaccines herein. Adjuvants known to those of ordinary skill in the art may be employed, such as those described in Vogel et al, "A Complex of Vaccine Adjuvants and Excipients" (2 nd edition), which is incorporated herein by reference in its entirety. Examples of known adjuvants include, but are not limited to: complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide adjuvant, Lipopolysaccharide (LPS), RIBI adjuvant, MF-59, etc.

The vaccine compositions herein may further comprise pharmaceutically acceptable carriers, diluents, preservatives, solubilizers, emulsifiers and like excipients. For example, pharmaceutically acceptable carriers are known and include, but are not limited to, water for injection, saline solution, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. Pharmaceutically acceptable carriers, diluents and other excipients can be found, for example, in Remington's pharmaceutical Sciences.

The vaccine compositions herein may be in a form suitable for systemic or local (especially in the respiratory tract) administration. Methods of administering vaccine compositions include, but are not limited to: intramuscular inoculation, intradermal inoculation, subcutaneous inoculation, nasal drops, aerosol inhalation, reproductive tract, rectal, oral, or any combination thereof.

In some embodiments, the vaccines herein prevent, eliminate or reduce a novel coronavirus infection or at least one symptom thereof in a subject, such as a respiratory symptom (e.g., nasal congestion, sore throat, hoarseness), headache, cough, sputum, fever, rales, wheezing, dyspnea, pneumonia due to infection, severe acute respiratory syndrome, renal failure, and the like.

Also contemplated herein is an immunoconjugate (also referred to as an immunoconjugate) comprising the immunogen herein and other materials coupled thereto. The additional substance may be a targeting substance (e.g., a moiety that specifically recognizes a particular target), a therapeutic substance (e.g., a drug, a toxin, a cytotoxic agent), a labeling substance (e.g., a fluorescent label, a radioisotope label).

Also provided in the present disclosure is a combination product comprising an immunogenic peptide, nucleotide molecule, vector, host cell and/or vaccine of the present disclosure, and may further comprise one or more other substances that contribute to better functioning or enhancing the stability of the aforementioned substances in preventing and/or treating a novel coronavirus infection or a symptom thereof. For example, the other substances may include other vaccines against coronavirus S or S1, such as S or S1 vaccines from including, but not limited to SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV; other active agents that benefit from T cell activation and/or memory immune response with T cells.

Immunization method

Also provided herein is a method for preventing and/or treating a novel coronavirus infection and/or symptoms thereof, comprising: administering at least once a prophylactically and/or therapeutically effective amount of one or more vaccines of the present disclosure. Inoculation regimes that may be used include, but are not limited to: systemic immunization modes, such as intramuscular injection, subcutaneous injection, intradermal injection and the like; and (3) immunization in respiratory tract, such as atomization, nasal drip and the like. In some embodiments, the primary immunization employs systemic or intrarespiratory vaccination, preferably systemic vaccination.

In some embodiments of the disclosure, the interval between each two vaccinations is at least 1 week, e.g., 2 weeks, 4 weeks, 2 months, 3 months, 6 months, or longer intervals.

In some embodiments, the primary immunization is performed with a DNA vaccine and the one or more booster immunizations are performed with a recombinant viral vaccine. The methods of immunization of the present disclosure may be by "prime-boost" or "prime-boost-re-boost", by a single systemic or local immunization of the respiratory tract, or by a combination of both immunization modalities.

Depending on the characteristics of the different vector vaccines, in some preferred embodiments, a systemic prime is performed using a recombinant DNA vaccine to establish a systemic immune response, followed by one or more boosts using other vaccines (e.g., recombinant adenoviral vaccines or recombinant poxvirus vaccines), which may include at least one boost in the respiratory tract (e.g., using an adenoviral vaccine).

The vaccine-specific immune response that can be effectively established in the local and systemic respiratory tract systems using the immunization methods herein helps to enhance the effectiveness of vaccine protection.

Providing the combination product herein in the form of a pharmaceutical pack or kit may, for example, be packaged in one or more containers, for example sealed containers such as ampoules or sachets, indicating the amount of composition, for example, one or more of the vaccine compositions herein or one or more of its ingredients. The vaccine compositions may be provided as a liquid, sterile lyophilized powder, or anhydrous concentrate, etc., which may be diluted, reconstituted and/or formulated with an appropriate liquid (e.g., water, saline, etc.) immediately prior to use to obtain the appropriate concentration and form for administration to a subject.

The combination product of the present disclosure can be used for the characteristic of locally inducing high-level antigen-specific CD8+ T cell responses in the respiratory tract, making it promising for the prevention and treatment of respiratory tract pathogen infection, reduction of respiratory tract pathogen pathogenicity, and respiratory tract tumors.

Examples

The disclosure is further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure. Appropriate modifications, variations and changes may be made by those skilled in the art to the present disclosure, which modifications and changes are within the scope of the present disclosure.

The experimental procedures for the conditions not specified in the examples below can be carried out by methods conventional in the art, for example, by referring to the molecular cloning, A Laboratory Manual, New York, Cold Spring Harbor Laboratory Press, 1989 or according to the conditions recommended by the supplier. Methods for sequencing DNA are conventional in the art and tests are also available from commercial companies.

Unless otherwise indicated, percentages and parts are by weight. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present disclosure. The preferred embodiments and materials described herein are intended to be exemplary only.

The experimental animals, immunization protocols, immunogens, pseudoviruses and detection methods involved in the following experiments were as follows:

I. experimental animals: 6-8 week-old female C57/BL6 mice

Immunization protocol: the left hind limb and the right hind limb of the mouse are respectively injected intramuscularly

Selection of immunogen:

the S and RBD sequences are from Genebank NC-045512.2, segment acid respiratory syndrome neutron virus 2 isocyanate Wuhan-Hu-1, complete genome; the hFn sequence is from Genebank M97164.1, the HA2 sequence is from Genebank AGI60292.1, and the specific sequences are shown in the sequence table.

1. Recombinant plasmid vaccine (DNA): pcDNA3.1-sRGB-hFn, pcDNA3.1-S, pcDNA3.1 (empty);

2. recombinant protein subunit vaccine (protein): RBD protein (not comprising disulfide modifications), sRBD-hFn protein;

3. recombinant human cell vector vaccine: k562, K562-S, K562-HA 2-sRGB.

Immunogen preparation and immunization dose:

preparation of immunogens see example 1

The immunogen immunizing doses used in the examples are as follows:

1. recombinant plasmid vaccine (DNA): 100 mu g/mouse, 100 mu L, dissolved in sterile physiological saline;

2. recombinant protein subunit vaccine (protein): the proteins (dissolved in sterile PBS) were mixed with aluminum adjuvant (aluminum, InvivoGen, cat # 5200) at a volume ratio of 1:1 and immunized with 10. mu.g/mouse of RBD protein, 100. mu.L; sRGB-hFn protein, 20. mu.g/mouse, 100. mu.L;

3. recombinant human cell vector vaccine (K562, K562-S, K562-HA 2-sRGB): 1E6 cells/mouse, 100. mu.L, dissolved in sterile PBS.

V. immunization interval:

specific immunization intervals are given in the following table

SARS-CoV2 envelope Pseudovirus (Pseudovirus) packaging:

1. 293T cells were prepared the day before transfection and used for transfection and expression of packaging plasmids. Cells were diluted to 5X 10 with DMEM complete medium⁶one/mL of cells, 1mL of diluted cells were plated in a 10cm dish at 37 ℃ with 5% CO₂Culturing overnight;

2. sucking SARS-CoV2 membrane protein plasmid pcDNA3.1-S4 μ g and pNL4-3 delta env skeleton plasmid 8 μ g (NIH AIDS Reagent Program,3418) into 500 μ L DMEM without double antibody (serum-free, double antibody is mixed solution of streptomycin), and incubating at room temperature for 5 min;

3. diluting 24 μ L TurboFect (Thermo Fisher Scientific) with DMEM-free medium to a final volume of 500 μ L/sample, and incubating at room temperature for 5 min;

4. the two samples 2 and 3 were mixed well, 1000. mu.L/final sample volume, incubated at room temperature for 20min, and added to 293T cells in a 10cm dish after incubation. After 6h, replacing fresh 15mL of complete culture medium, and continuously culturing in a cell culture box for 48 h;

5. after the culture is finished, collecting cell culture supernatant in a 10cm dish, centrifuging for 10min at 4000g and 4 ℃ in a 15mL centrifuge tube, filtering the cell culture supernatant into a new 15mL centrifuge tube by using a 0.45 mu m filter, freezing the cell culture supernatant at the temperature of minus 80 ℃, and titrating the cell culture supernatant for later use.

Construction of 293T cells stably expressing hACE2 receptor:

1. an artificial synthetic human ACE2(hACE2) sequence (Genebank # NCBI _ NP-001358344.1) as shown in SEQ ID NO:13 with an Age1 cleavage site at the 5 'end and an Xba1 cleavage site at the 3' end, the synthetic fragment cleaved with vector plasmid pHAGE-MCS-puro using Age1 (Thermo Scientific, cat # FD1464) with Xba1 (Thermo Scientific, cat # FD0685) and recovered by gel electrophoresis followed by gel cutting and recovery of the cleaved fragments using Sanprep column DNA gel recovery kit (Promega, cat # A9282).

2. The gene recovery product was ligated to the digested linearized vector using T4 DNA ligase (Thermo Scientific Co., Ltd., cat # 2011A): coli Stable was transformed with the ligation product and grown overnight on ampicillin-containing plates. On day 2, single colonies were randomly picked and sequenced, the mutation sites were corrected, and after confirming that the entire sequence was correct, a lentiviral expression plasmid (pHAGE-hACE2-puro) for the hACE2 gene was successfully cloned.

3. 10cm dishes were taken and inoculated about 5X 10 in each dish⁶293T cells, which ensures that the cell density reaches 90% when the transfection is carried out on the next day; three plasmids, namely pHAGE-hACE2-puro, a lentivirus packaging plasmid psPAX and VSVG are added according to the mass ratio of 1: 2: 1 ratio to transfect 293T cells.

Culturing at 4.37 deg.C in 5% incubator for about 48 hr, and collecting cell supernatant according to cell condition. The collected cell supernatant was filtered through a 0.45 μm filter and concentrated with PEG 8000 to obtain a purified hACE2 lentivirus.

5. Spread 5X 10 one day in advance⁵The 293T cells of (1) were put into one well of a 12-well plate, and 500. mu.L of the virus concentrated in step 2, 1000g, was added to the plated cells the next day, followed by centrifugation for 2 hours.

6. After completion of the centrifugal infection, the cells were further cultured at 37 ℃ for about 12 hours in a 5% incubator, the medium was changed to a cell culture medium supplemented with 1. mu.g/mL puromycin (puro), and the 293T cells which had the hACE2 gene integrated therein were finally survived, and 293T cells (capable of binding to S protein) which stably expressed hACE2 were selected by flow screening.

VIII, detection method:

blood collection:

and (3) after the last immunization is finished and at the 4 th week, taking the mouse off the neck and before death, collecting the whole blood at the periphery of the mouse by an eyeball picking method, collecting the whole blood in a 1.5mL EP tube, standing at room temperature to enable the whole blood to be coagulated naturally, centrifuging the coagulated mouse serum at 7000g for 15 min. Mouse sera were transferred to new 1.5mL EP tubes. Samples were inactivated at 56 ℃ for 30min prior to the experiment to destroy complement activity in the serum. And the tube is centrifuged for a short time before inactivation, so that the residual samples on the tube wall and the bottle cap are avoided. The bath level should be below the sample level but not above the cap.

ELSIA detection of bound antibodies:

1. the detected antigenic protein (S1 or RBD) was diluted with a 4 ℃ pre-chilled ELISA coating to a final concentration of 1. mu.g/mL. Add 100. mu.L of the coating antigen solution to each well of the ELISA plate, and keep it at 4 ℃ overnight;

2. the next day, the ELISA plate was removed, the coating solution was discarded, and the plate was washed 3 times with 0.05% PBST buffer, 220. mu.L each time;

3. after washing, patting the mixture on absorbent paper, sealing each hole by using 200 mu L of ELISA sealing solution (0.5% skimmed milk powder, dissolved in PBST), and sealing at room temperature for 2 h;

4. after blocking, wash the plate 3 times with 0.05% PBST, 220 μ L each time;

5. for serum or plasma, dilutions were performed with ELISA sample dilutions (0.5% skim milk powder, PBST lysis) starting from 1:100 and 2-fold dilutions were performed. A negative control was set with non-immunized mouse serum. Setting Blank holes, only adding sample diluent, preparing 2 multiple holes for each sample, wherein the final volume of each hole is 100 mu L, and incubating for 3h at room temperature;

6. after the incubation of the sample is finished, the plate is continuously washed by PBST for 5 times, and each time is 220 mu L;

7. diluting the corresponding proportion of secondary antibody (goat anti-mouse) with ELISA blocking solution (0.5% skimmed milk powder, PBST dissolved), adding 100 μ L per well, and incubating at room temperature for 1-1.5 h;

8. after the secondary antibody incubation was completed, the plate was washed 5 times with 0.05% PBST, 220 μ L each time;

9. dissolving a pair of gold and silver sheet OPD substrates in 20mL of deionized water, then adding 100 mu L of gold and silver sheet OPD substrates into each hole, and reacting for 5min in a dark place;

10. after the color development was completed, 50. mu.L of 2nM H was used₂SO₄The termination is performed and the OD is read on the microplate reader₄₉₂-OD₆₃₀A value;

11. the antibody titer was determined as the reciprocal of the serum dilution ratio corresponding to a value (negative mean + SD) at which the OD492 of the last dilution was greater than 2-fold.

293T-ACE2 cells detect neutralizing antibodies:

1. a96-well transparent bottom blackboard is taken for carrying out a neutralization experiment, a Cell Control (CC) (150 mu L) is arranged in the first column, a Virus Control (VC) (100 mu L) is arranged in the second column, all the other wells are sample wells, and serum samples are diluted in a multiple proportion, so that the volume in the final wells is 100 mu L.

2. In addition to the cell control group, 50. mu.L of SARS-CoV-2 pseudovirus diluent was added to each well, so that each well finally contained pseudovirus at 200TCID₅₀。

3. Gently shaking and mixing, placing the blackboard with 96 holes bottom in a cell culture box, keeping the temperature at 37 ℃ and 5% CO₂Incubate for 1 h.

4. When the incubation time reached 20min, 293T-hACE2 target cells were initially prepared and diluted to 10 with complete medium⁵Individual cells/mL.

5. When the incubation time is up to 1h, 100. mu.L of target cells are added to each well of a 96-well transparent bottom blackboard, so that the cells in each well are 10⁴And (4) respectively.

6. Gently shaking the 96-well transparent bottom blackboard all around to uniformly disperse the cells in the holes, and then placing the blackboard in a cell culture box at 37 ℃ and 5% CO₂Culturing for 48 h.

7. Culturing for 48h, taking out 96-well transparent bottom blackboard from the cell culture box, sucking off supernatant in the wells, adding 100 μ L PBS to each well for washing, sucking off PBS, adding 50 μ L of 1 × lysis buffer (from Cat # E153A of Promega corporation) to each well, and incubating on a horizontal shaker at room temperature for 30min to fully lyse the cells;

8. add 30. mu.L of luciferase substrate (available from Promega, Cat # E1501) to a 96-well blackboard and use the instrument

The luciferase activity is detected by a 96-microplate luminescence-detection instrument.

9. Reading values of fluorescein are derived, neutralization inhibition rates are calculated, and ID is calculated by utilizing Graphpad Prism 5.0 software according to the results of the neutralization inhibition rates₅₀。

Example 1: construction of S and sRBD-hFn (disulfide bond modification) eukaryotic expression vector and protein expression

To study the function of RBD, eukaryotic expression vectors for S and sRBD-hFn were constructed and the mature protein was expressed in vitro in a cell line.

First, we artificially synthesized the S gene (SEQ ID NO:11 in sequence), the sRBD-hFn gene (SEQ ID NO:7 in sequence, in which the sRBD part is based on the sequence shown in SEQ ID NO:3 and hFn part is based on the sequence shown in SEQ ID NO: 5), the synthesized fragment and the vector plasmid pcDNA3.1 (available from Youbao) were digested with BamHI (Thermo Scientific, FD0054) and Not1 (Thermo Scientific, FD0596) and recovered by gel electrophoresis followed by gel cutting, and the digested fragments were recovered using Sanprep column DNA gel recovery kit (Promega, cat # A9282). The sRGB-hFn gene has cysteine added to the N and C ends of RBD part to form disulfide bond at the root of RBD and stabilize the structure of RBD. sRGB was expressed at the N-terminus of hFn, and the two were ligated using a linker.

Subsequently, the gene recovery product was ligated with the digested linearized vector by the method of T4 DNA ligase (Thermo Scientific Co., Ltd., cat. No. 2011A): coli Stable was transformed with the ligation product and grown overnight on ampicillin-containing plates. On day 2, single colonies were randomly selected for sequencing, mutation site correction, and after all sequences were verified to be correct, eukaryotic expression vectors pcDNA3.1-S and pcDNA3.1-sRBD-hFn of S and sRBD-hFn were successfully cloned, and the plasmid construction map is shown in FIG. 1A.

We further examined whether S and sRBD-hFn could be expressed in the eukaryotic cell line 293T.

First, 6-well plates were prepared and seeded at approximately 6X 10 in each well⁵293T cells, which ensures that the cell density reaches 90% when the transfection is carried out on the next day; 293T cells (transfection reagent TurboFect) were transfected with pcDNA3.1, pcDNA3.1-S and pcDNA3.1-sRGB-hFn, respectively. Culturing at 37 deg.C for about 48 hr in 5% incubator, collecting cells according to cell condition, and performing protein immune printingThe trace (WB) identification shows that S is expressed in cells and has the size of about 200 KD; sRGB-hFn was expressed in the cells at around 55kD, whereas pcDNA3.1 control transfected cells were unable to detect expression of the sRGB-hFn protein (FIG. 1B).

The structure of the expressed sRGB-hFn is: sRBD expression at the N-terminus of hFn, employed between (G4S)₃And (4) connecting.

Example 2: construction of lentivirus expression vectors pHAGE-S-puro and pHAGE-sRBD-HA2-puro and expression verification of S protein and sRBD-HA2 displayed on K562 cell membrane

In order to verify whether the conformation of the disulfide bond modified RBD (sRBD) is correct, lentiviral expression vectors pHAGE-S-puro and pHAGE-sRBD-HA2-puro are constructed, and the protein is expressed on K562 cell membranes, so that the expression and conformation of the protein are verified.

First, we artificially synthesized the S gene (SEQ ID NO:11 in sequence), the sRBD-HA2 gene (SEQ ID NO:9 in sequence), the synthesized fragment with a Not1 cleavage site at the 5 'end and an Xba1 cleavage site at the 3' end, and the synthesized fragment cleaved with the vector plasmid pHAGE-MCS-puro (available from Shanghai Xinwan Biotechnology Co., Ltd.) using Not1 (Thermo Scientific Co., FD0596) and Xba1 (Thermo Scientific Co., FD0685) and recovered the cleaved fragments after gel electrophoresis using a Sanprep column DNA gel recovery kit (Promega Co., Ltd., Cat. A9282).

The gene recovery product was ligated to the digested linearized vector using T4 DNA ligase (Thermo Scientific Co., Ltd., cat # 2011A): coli Stable was transformed with the ligation product and grown overnight on ampicillin-containing plates. On day 2, single colonies were randomly picked for sequencing, mutation site correction, and after all sequences were verified to be correct, lentiviral expression vectors pHAGE-S-puro and pHAGE-sRBD-HA2-puro of S and sRBD-HA2 were successfully cloned (FIG. 2A).

Transfecting 293T cells with pHAGE-S-puro and pHAGE-sRBD-HA2-puro, and identifying the expression of protein in K562-S cells by Western blotting (FIG. 2B); meanwhile, flow cytometric staining identification is carried out, and the S protein and the sRBD-HA2 protein are found to be expressed on cell membranes and can be combined with a receptor ACE2, while the expression of related proteins cannot be detected by cells infected with a control, so that the conformation of the disulfide bond modified RBD is kept relatively complete, and the main function of the disulfide bond modified RBD is not influenced (FIG. 2C).

The experimental procedure was as follows: 6-well plates were taken and seeded at approximately 6X 10 in each well⁵293T cells, which ensures that the cell density reaches 90% when the transfection is carried out on the next day; 293T cells were transfected with pHAGE-S-puro and pHAGE-sRBD-HA2-puro (transfection reagent TurboFect). Culturing at 37 deg.C for about 48 hr in 5% incubator, collecting cells according to cell condition, performing Western Blotting (WB) identification, and finding that S is expressed in cells with size of about 200 KD; sRGB-HA 2 was expressed in the cells at around 80kD, whereas the pCDNA3.1 control transfected cells were unable to detect expression of the sRGB-hFn protein (FIG. 2B). Preparation of 5X 10⁵K562 cells, resuspended in 500 μ L complete cell culture medium, placed in one well of a 12-well plate. Concentrated lentivirus was added to the plated cells at 1000g and centrifuged for 2 hours. After the completion of the centrifugal infection, the cells were further incubated at 37 ℃ in a 5% incubator for about 48 hours. After the infection, the K562 cells were cultured in RPMI (10% FBS) having a puromycin concentration of 4. mu.g/ml because the expression vector plasmid was resistant to puromycin, and the cells which could survive last were S gene-integrated and sRBD-HA 2-integrated cells. The infected cells were then used to detect S protein expression by Western blotting, using ACE2-C-AVI-6his as the primary antibody (Shanghai offshore science and technology Co., Ltd., model 0331753-. The results showed that high expression of the target protein could be detected by western blotting (fig. 2B).

The flow staining method was performed by indirect staining using ACE2-C-AVI-6his (Shanghai near-shore technologies Co., Ltd., model 0331753-. The results show that, after continuous enrichment, more than 80% of K562-S cells and K562-HA 2-sRGB cells were able to bind ACE2, indicating that the conformation of disulfide-modified RBD itself does not affect its primary function (FIG. 2C).

Example 3: induction effects of RBD proteins on mouse binding and neutralizing antibodies

The immunization combination was evaluated to induce the titer of binding antibodies against RBD protein and S1 protein 2 weeks after immunization of C57/BL6 mice with RBD protein (purchased from tokensry biotechnology from south kyo) with aluminum adjuvant as adjuvant. At the same time, 4 weeks after completion of immunization, the neutralizing antibody titer against SARS-CoV-2 pseudovirus was evaluated.

The experimental procedure was as follows: mice were randomly divided into 2 groups, and designated as a control group and an RBD group, respectively, based on the immunogen. Specific immunological combinations are shown in table 1. The RBD group produced binding antibody titers against the RBD protein and the S1 protein as shown in fig. 3A: the titers of the binding antibodies against the different proteins (S1 protein and RBD protein) were substantially identical, with a mean value of about 8000, and some of the mouse binding antibodies were not induced, probably due to the individual differences of the mice. 4 weeks after immunization, the RBD group produced neutralizing antibody titers against SARS-Cov-2 pseudovirus as shown in FIG. 3B: except for one titer above 1000, and others around 200, some of the mouse neutralizing antibodies were not induced, probably due to individual differences in mice.

This experiment demonstrates that RBD protein induces the production of bound antibodies (similar to S protein induction) and that pure RBD protein activates higher levels of neutralizing antibodies.

TABLE 1 RBD protein induced binding antibody and neutralizing antibody assay

Example 4: induction effect of plasmid pcDNA3.1-sRBD-hFn on mouse binding antibody and neutralizing antibody

Based on the conclusion made in example 1 (that the plasmid was able to correctly express the desired protein), C57/BL6 mice were immunized with the disulfide-modified RBD plasmid (pcDNA3.1-sRBD-hFn), and the combination of the immunization was evaluated to induce a binding antibody titer against the RBD protein and a neutralizing antibody titer against the SARS-CoV-2 pseudovirus 2 weeks after completion of the immunization.

The experimental procedure was as follows: mice were randomly divided into 2 groups, and designated as a control group and an RBD-hFn group, respectively, based on the immunogen of the second needle. Specific immunological combinations are shown in table 2. The different immunization combinations produced binding antibody titers against the RBD protein as shown in figure 4A: most are around 2000. 2 weeks after the end of the second needle immunization, the different immunization combinations produced neutralizing antibody titers against SARS-CoV-2 pseudovirus as shown in FIG. 4B: the neutralizing antibody titers were around 20, except one was greater than 80.

The experiment proves that the modified RBD recombinant plasmid vaccine can induce neutralizing antibodies in mice.

TABLE 2 modified RBD-hFn plasmid induced binding antibody and neutralizing antibody assay

Example 5: effect of K562-HA2-sRBD cells and K562-S cells on inducing mouse-bound antibodies and neutralizing antibodies

According to the results obtained in example 4, 10 mice of the sRGB-hFn group were divided into two groups, and K562-S and K562-HA 2-sRGB were re-immunized, and the combination of immunization was evaluated to induce a binding antibody titer against RBD protein and a neutralizing antibody titer against SARS-CoV-2 pseudovirus 2 after completion of immunization for 2 weeks.

Specific immunological combinations are shown in table 3. 2 weeks after the end of immunization, the different immunization combinations produced a binding antibody titer against the RBD protein as shown in fig. 5A: the K562-S group bound antibody titers were mostly around 50000, and the K562-HA2-sRBD group bound antibody titers were mostly around 80000. 2 weeks after immunization, different immunization combinations produced neutralizing antibody titers against SARS-CoV-2 pseudovirus as shown in FIG. 5B: the neutralizing antibody titer of the K562-S group is mostly about 350, one neutralizing antibody titer of the K562-HA 2-sRGB group reaches 2000, and the other neutralizing antibody titer of the K562-HA 2-sRGB group is about 400.

This experiment demonstrates that K562-HA2-sRBD cells are able to induce relatively higher binding and neutralizing antibodies in mice than K562-S cells.

TABLE 3K 562-S and K562-HA 2-sRGB Induction binding antibody and neutralizing antibody experiments

Example 6: effect of sRGB-hFn protein on inducing mouse-binding antibody and neutralizing antibody

C57/BL6 mice were immunized with the purified sRBD-hFn protein, and the combination was evaluated to induce a binding antibody titer against RBD protein and a neutralizing antibody titer against SARS-CoV-2 pseudovirus 1 week after completion of the immunization.

The experimental procedure was as follows: mice were randomly divided into 2 groups, designated control and sRGB-hFn groups, respectively, based on the immunogen. Specific immunological combinations are shown in table 4. One week after completion of immunization, the titers of binding antibodies generated against RBD protein are shown in fig. 6A: the sRBD-hFn group had three mice with binding antibodies reaching 52000, all but one at 3200, and more than ten thousand. 1 week after the end of immunization, neutralizing antibody titers against SARS-CoV-2 pseudovirus were generated as shown in FIG. 6B: there were three neutralizing antibodies, all with titers of over ten.

TABLE 4 sRBD-hFn protein induced binding antibody and neutralizing antibody assay

All documents referred to in this disclosure are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications to the disclosure may be made by those skilled in the art after reading the above teachings of the disclosure, and such equivalents may fall within the scope of the disclosure as defined by the appended claims.

Attached Table 1. sequence Listing correspondence information

Sequence listing

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tgctatggcg tgagccccac aaagctgaat gacctgtgct ttaccaacgt gtacgccgat 240

tccttcgtga tcaggggcga cgaggtgcgc cagatcgcac caggacagac aggcaagatc 300

gcagactaca attataagct gcctgacgat ttcaccggct gcgtgatcgc ctggaactct 360

aacaatctgg atagcaaagt gggcggcaac tacaattatc tgtaccggct gtttagaaag 420

tctaatctga agccattcga gagggacatc tccacagaaa tctaccaggc cggctctacc 480

ccctgcaatg gcgtggaggg ctttaactgt tatttccctc tgcagagcta cggcttccag 540

ccaacaaacg gcgtgggcta tcagccctac cgcgtggtgg tgctgtcttt tgagctgctg 600

cacgcacctg caacagtgtg cggaccaaag aagagcacca atctggtgaa gaacaagtgc 660

gtgaacttca acttcaacgg actgaccggc acaggcgtgc tgaccgagtc caacaagaag 720

ttcctgcctt ttcagcagtt cggcagggac atcgcagata ccacagacgc cgtgcgcgac 780

cctcagaccc tggagatcct ggatatcaca ccatgc 816

<210> 4

<211> 272

<212> PRT

<213> Artificial sequence

<400> 4

Met Val Cys Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn

210 215 220

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys

225 230 235 240

Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp

245 250 255

Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys

260 265 270

<210> 5

<211> 549

<212> DNA

<213> Artificial sequence

<400> 5

atgacgaccg cgtccacctc gcaggtgcgc cagaactacc accaggactc agaggccgcc 60

atcaaccgcc agatcaacct ggagctctac gcctcctacg tttacctgtc catgtcttac 120

tactttgacc gcgatgatgt ggctttgaag aactttgcca aatactttct tcaccaatct 180

catgaggaga gggaacatgc tgagaaactg atgaagctgc agaaccaacg aggtggccga 240

atcttccttc aggatatcaa gaaaccagac tgtgatgact gggagagcgg gctgaatgca 300

atggagtgtg cattacattt ggaaaaaaat gtgaatcagt cactactgga actgcacaaa 360

ctggccactg acaaaaatga cccccatttg tgtgacttca ttgagacaca ttacctgaat 420

gagcaggtga aagccatcaa agaattgggt gaccacgtga ccaacttgcg caagatggga 480

gcgcccgaat ctggcttggc ggaatatctc tttgacaagc acaccctggg agacagtgat 540

aatgaaagc 549

<210> 6

<211> 183

<212> PRT

<213> Artificial sequence

<400> 6

Met Thr Thr Ala Ser Thr Ser Gln Val Arg Gln Asn Tyr His Gln Asp

1 5 10 15

Ser Glu Ala Ala Ile Asn Arg Gln Ile Asn Leu Glu Leu Tyr Ala Ser

20 25 30

Tyr Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala

35 40 45

Leu Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg

50 55 60

Glu His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg

65 70 75 80

Ile Phe Leu Gln Asp Ile Lys Lys Pro Asp Cys Asp Asp Trp Glu Ser

85 90 95

Gly Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Asn Val Asn

100 105 110

Gln Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro

115 120 125

His Leu Cys Asp Phe Ile Glu Thr His Tyr Leu Asn Glu Gln Val Lys

130 135 140

Ala Ile Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg Lys Met Gly

145 150 155 160

Ala Pro Glu Ser Gly Leu Ala Glu Tyr Leu Phe Asp Lys His Thr Leu

165 170 175

Gly Asp Ser Asp Asn Glu Ser

180

<210> 7

<211> 1470

<212> DNA

<213> Artificial sequence

<400> 7

atggccctcc ccgtgacagc cctgctcctg cctctcgccc tgctgctcca cgccgccaga 60

ccagtgtgcc ctacagagtc tattgtgcgc ttcccaaaca tcacaaacct gtgccctttc 120

ggcgaggtgt tcaacgccac acggttcgcc tctgtgtacg cctggaaccg gaagcggatt 180

tctaactgcg tggccgacta ctctgtgctg tacaacagcg cctctttctc tacattcaag 240

tgctacggcg tgtctccaac aaagctcaac gacctgtgct tcacaaacgt gtacgccgac 300

tctttcgtga ttagaggcga cgaggtgaga cagattgccc caggccagac cggcaagatt 360

gccgactaca actacaagct ccccgacgac ttcacaggct gcgtgattgc ctggaactct 420

aacaacctcg actctaaggt gggcggcaac tacaactacc tgtacaggct gttccggaag 480

tctaacctga agcctttcga gcgggacatt agcaccgaga tttaccaggc cggctctacc 540

ccttgcaacg gcgtggaggg cttcaactgc tacttcccat tgcagtctta cggcttccag 600

cctacaaacg gcgtgggcta ccagccttac cgggtggtgg tgctgtcttt cgagctgctc 660

cacgcccccg ccacagtgtg cggcccaaag aagtctacaa acctggtgaa gaacaagtgc 720

gtgaacttca acttcaacgg cctcacaggc acaggcgtgc tcaccgagtc taacaagaag 780

ttcctgcctt tccagcagtt cggcagggac attgccgaca ccaccgacgc cgtgagggac 840

cctcagacac tagagattct cgacatcacc ccatgctctg gcggcggcgg ctctggcggc 900

ggcggctctg gcggcggcgg cagcaccaca gcctctacat ctcaggtgag gcagaactac 960

caccaggact ctgaggccgc cattaacagg cagattaacc tggaactgta cgcctcttac 1020

gtgtacctgt ctatgtctta ctacttcgac cgggacgacg tggccctgaa gaacttcgcc 1080

aagtacttcc tccaccagtc tcacgaggag agggagcacg ccgagaagct gatgaagctc 1140

caaaaccagc ggggaggccg gattttctta caggacatta agaagcccga ctgcgacgac 1200

tgggagtctg gcctgaacgc gatggagtgc gccctccact tggagaagaa cgtgaaccag 1260

tccctgctgg aactacacaa gctcgccacc gacaagaacg accctcacct gtgcgacttc 1320

attgagaccc actacctcaa cgagcaggtg aaggccatta aagaactcgg cgaccacgtc 1380

acaaacctga gaaagatggg cgcccccgag tccggcctcg ccgagtacct gttcgacaag 1440

cacacactgg gcgactctga caacgagtct 1470

<210> 8

<211> 490

<212> PRT

<213> Artificial sequence

<400> 8

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Val Cys Pro Thr Glu Ser Ile Val Arg Phe Pro

20 25 30

Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg

35 40 45

Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val

50 55 60

Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys

65 70 75 80

Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn

85 90 95

Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile

100 105 110

Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro

115 120 125

Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp

130 135 140

Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys

145 150 155 160

Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln

165 170 175

Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe

180 185 190

Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln

195 200 205

Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala

210 215 220

Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys

225 230 235 240

Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu

245 250 255

Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala

260 265 270

Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp

275 280 285

Ile Thr Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Ser Thr Thr Ala Ser Thr Ser Gln Val Arg Gln Asn Tyr

305 310 315 320

His Gln Asp Ser Glu Ala Ala Ile Asn Arg Gln Ile Asn Leu Glu Leu

325 330 335

Tyr Ala Ser Tyr Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp Arg Asp

340 345 350

Asp Val Ala Leu Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His

355 360 365

Glu Glu Arg Glu His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg

370 375 380

Gly Gly Arg Ile Phe Leu Gln Asp Ile Lys Lys Pro Asp Cys Asp Asp

385 390 395 400

Trp Glu Ser Gly Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys

405 410 415

Asn Val Asn Gln Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys

420 425 430

Asn Asp Pro His Leu Cys Asp Phe Ile Glu Thr His Tyr Leu Asn Glu

435 440 445

Gln Val Lys Ala Ile Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg

450 455 460

Lys Met Gly Ala Pro Glu Ser Gly Leu Ala Glu Tyr Leu Phe Asp Lys

465 470 475 480

His Thr Leu Gly Asp Ser Asp Asn Glu Ser

485 490

<210> 9

<211> 1806

<212> DNA

<213> Artificial sequence

<400> 9

atgaaaacaa ttattgccct gtcttacatt ttctgcctcg tgttcgccga ctacaaggac 60

gacgacgaca agtccctgca ggtgtgccct accgagtcta tcgtgcgctt cccaaacatc 120

acaaacctgt gccctttcgg cgaggtgttc aacgccaccc ggttcgcctc tgtgtacgcc 180

tggaaccgga agcggatttc taactgcgtg gccgactact ccgtgctgta caactctgcc 240

tctttctcca cattcaagtg ctacggcgtg tcccctacca agctgaacga cctgtgcttc 300

accaacgtgt acgccgactc tttcgtgatt aggggcgacg aggtgagaca gattgcccct 360

ggccagacag gcaagatcgc cgactacaac tacaagctcc ctgacgactt cacaggctgc 420

gtgattgcct ggaactctaa caacctggac tctaaggtgg gcggcaacta caactacctg 480

tacagactgt tccggaagtc taacctcaag ccattcgagc gcgacattag caccgagatt 540

taccaggccg gcagcacccc atgcaacggc gtggagggct tcaactgcta cttcccactt 600

caatcttacg gcttccagcc aacaaacggc gtgggctacc agccataccg ggtggtggtg 660

ctgtccttcg agctactcca cgccccagcc acagtgtgcg gcccaaagaa gagcaccaac 720

ctcgtgaaga acaagtgcgt gaacttcaac ttcaacggcc tgacaggcac aggcgtgctc 780

accgagtcta acaagaagtt cctccctttc cagcagttcg gcagggacat cgccgacacc 840

accgacgccg tgcgcgaccc tcagacactc gaaattctgg acatcacccc ttgctctggc 900

ggcggcggct ctggcctgtt cggcgccatt gccggcttca tcgagaacgg ctgggagggc 960

ctcattgacg gctggtacgg cttcaggcac cagaacgccc agggcgaggg cacagccgcc 1020

gactacaagt ccacccagtc cgccattgac cagatcacag gcaagctgaa cagactcatt 1080

gaaaaaacaa accagcagtt cgagctgatt gacaacgagt tcaacgaggt ggagaagcag 1140

attggcaacg tgattaactg gacacgggac tctattacag aggtgtggtc ttacaacgcc 1200

gagttactcg tggcaatgga gaaccagcac acaattgacc tcgccgactc tgagatggac 1260

aagctgtacg agcgggtgaa gagacagctc agagagaacg ccgaggagga cggcacaggc 1320

tgcttcgaga tattccacaa gtgcgacgac gactgcatgg ccagcatccg gaacaacaca 1380

tacgaccact ctaagtaccg ggaggaggcc atgcagaacc gcatccagat tgacccagtg 1440

aagctgtcta gcggctacaa ggacgtggct agcaccacca cacccgcccc tagacctcct 1500

acccccgccc caacaattgc ctctcagcca ctgtctctca gacctgaggc gtgcaggccc 1560

gccgccggcg gcgccgtgca cacacggggc ctcgacttcg cctgcgacat ttacatttgg 1620

gccccactcg ccggcacatg cggcgtgctc ctcctgtctc tggtgatcac actgtactgc 1680

aagcggggca gaaagaagct cctgtacatt ttcaagcagc ctttcatgag acccgtgcag 1740

accacccagg aggaggacgg ctgctcttgc aggttccctg aggaggagga gggcggctgc 1800

gaacta 1806

<210> 10

<211> 602

<212> PRT

<213> Artificial sequence

<400> 10

Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Phe Ala

1 5 10 15

Asp Tyr Lys Asp Asp Asp Asp Lys Ser Leu Gln Val Cys Pro Thr Glu

20 25 30

Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu

35 40 45

Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys

50 55 60

Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala

65 70 75 80

Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn

85 90 95

Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly

100 105 110

Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp

115 120 125

Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp

130 135 140

Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu

145 150 155 160

Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile

165 170 175

Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu

180 185 190

Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr

195 200 205

Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu

210 215 220

Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn

225 230 235 240

Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly

245 250 255

Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln

260 265 270

Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro Gln

275 280 285

Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Gly Gly Gly Gly Ser

290 295 300

Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly

305 310 315 320

Leu Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ala Gln Gly Glu

325 330 335

Gly Thr Ala Ala Asp Tyr Lys Ser Thr Gln Ser Ala Ile Asp Gln Ile

340 345 350

Thr Gly Lys Leu Asn Arg Leu Ile Glu Lys Thr Asn Gln Gln Phe Glu

355 360 365

Leu Ile Asp Asn Glu Phe Asn Glu Val Glu Lys Gln Ile Gly Asn Val

370 375 380

Ile Asn Trp Thr Arg Asp Ser Ile Thr Glu Val Trp Ser Tyr Asn Ala

385 390 395 400

Glu Leu Leu Val Ala Met Glu Asn Gln His Thr Ile Asp Leu Ala Asp

405 410 415

Ser Glu Met Asp Lys Leu Tyr Glu Arg Val Lys Arg Gln Leu Arg Glu

420 425 430

Asn Ala Glu Glu Asp Gly Thr Gly Cys Phe Glu Ile Phe His Lys Cys

435 440 445

Asp Asp Asp Cys Met Ala Ser Ile Arg Asn Asn Thr Tyr Asp His Ser

450 455 460

Lys Tyr Arg Glu Glu Ala Met Gln Asn Arg Ile Gln Ile Asp Pro Val

465 470 475 480

Lys Leu Ser Ser Gly Tyr Lys Asp Val Ala Ser Thr Thr Thr Pro Ala

485 490 495

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

500 505 510

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

515 520 525

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

530 535 540

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

545 550 555 560

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

565 570 575

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

580 585 590

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

595 600

<210> 11

<211> 3843

<212> DNA

<213> novel coronavirus (SARS-CoV-2 virus)

<400> 11

atgttcgtgt ttctggtgct gctgcctctg gtgagctccc agtgcgtgaa cctgaccaca 60

aggacccagc tgccccctgc ctataccaat tccttcacac ggggcgtgta ctatcccgac 120

aaggtgttcc ggagcagcgt gctgcactcc acacaggatc tgtttctgcc tttcttttct 180

aacgtgacct ggttccacgc catccacgtg agcggcacca atggcacaaa gcggttcgac 240

aatccagtgc tgccctttaa cgatggcgtg tacttcgcct ccaccgagaa gtctaacatc 300

atcagaggct ggatctttgg caccacactg gacagcaaga cacagtccct gctgatcgtg 360

aacaatgcca ccaacgtggt catcaaggtg tgcgagttcc agttttgtaa tgatccattc 420

ctgggcgtgt actatcacaa gaacaataag tcttggatgg agagcgagtt tcgcgtgtat 480

tcctctgcca acaattgcac atttgagtac gtgtcccagc ccttcctgat ggacctggag 540

ggcaagcagg gcaatttcaa gaacctgagg gagttcgtgt ttaagaatat cgatggctac 600

ttcaaaatct actccaagca caccccaatc aacctggtgc gcgacctgcc acagggcttc 660

tctgccctgg agccactggt ggatctgccc atcggcatca acatcacccg gtttcagaca 720

ctgctggccc tgcacagaag ctacctgaca ccaggcgaca gctcctctgg atggaccgca 780

ggagcagcag cctactatgt gggctatctg cagcccagga ccttcctgct gaagtacaac 840

gagaatggca ccatcacaga cgccgtggat tgcgccctgg atcccctgtc tgagaccaag 900

tgtacactga agagctttac cgtggagaag ggcatctatc agacaagcaa tttcagggtg 960

cagcctaccg agtccatcgt gcgctttccc aatatcacaa acctgtgccc ttttggcgag 1020

gtgttcaacg caacccgctt cgcaagcgtg tacgcctgga ataggaagcg catctccaac 1080

tgcgtggccg actattctgt gctgtacaac agcgcctcct tctctacctt taagtgctat 1140

ggcgtgagcc ccacaaagct gaatgacctg tgctttacca acgtgtacgc cgattccttc 1200

gtgatcaggg gcgacgaggt gcgccagatc gcaccaggac agacaggcaa gatcgcagac 1260

tacaattata agctgcctga cgatttcacc ggctgcgtga tcgcctggaa ctctaacaat 1320

ctggatagca aagtgggcgg caactacaat tatctgtacc ggctgtttag aaagtctaat 1380

ctgaagccat tcgagaggga catctccaca gaaatctacc aggccggctc taccccctgc 1440

aatggcgtgg agggctttaa ctgttatttc cctctgcaga gctacggctt ccagccaaca 1500

aacggcgtgg gctatcagcc ctaccgcgtg gtggtgctgt cttttgagct gctgcacgca 1560

cctgcaacag tgtgcggacc aaagaagagc accaatctgg tgaagaacaa gtgcgtgaac 1620

ttcaacttca acggactgac cggcacaggc gtgctgaccg agtccaacaa gaagttcctg 1680

ccttttcagc agttcggcag ggacatcgca gataccacag acgccgtgcg cgaccctcag 1740

accctggaga tcctggatat cacaccatgc tccttcggcg gcgtgtctgt gatcacacca 1800

ggcaccaata caagcaacca ggtggccgtg ctgtatcagg acgtgaattg taccgaggtg 1860

cccgtggcaa tccacgcaga tcagctgacc cctacatggc gggtgtactc taccggcagc 1920

aacgtgttcc agacaagagc aggatgcctg atcggagcag agcacgtgaa caatagctat 1980

gagtgcgaca tccctatcgg cgccggcatc tgtgcctcct accagaccca gacaaactcc 2040

ccaaggagag cacggtctgt ggcaagccag tccatcatcg cctataccat gagcctgggc 2100

gccgagaatt ccgtggccta ctccaacaat tctatcgcca tccctaccaa cttcacaatc 2160

tccgtgacca cagagatcct gccagtgagc atgaccaaga catccgtgga ctgcacaatg 2220

tatatctgtg gcgattccac cgagtgctct aacctgctgc tgcagtacgg ctctttttgt 2280

acccagctga atagagccct gacaggcatc gccgtggagc aggacaagaa cacacaggag 2340

gtgttcgccc aggtgaagca aatctacaag accccaccca tcaaggactt tggcggcttc 2400

aacttcagcc agatcctgcc cgatcctagc aagccatcca agcggtcttt tatcgaggac 2460

ctgctgttca acaaggtgac cctggccgat gccggcttca tcaagcagta tggcgattgc 2520

ctgggcgaca tcgccgccag agacctgatc tgtgcccaga agtttaatgg cctgaccgtg 2580

ctgcctccac tgctgacaga tgagatgatc gcccagtaca catctgccct gctggcaggc 2640

accatcacaa gcggatggac cttcggcgca ggagccgccc tgcagatccc ctttgccatg 2700

cagatggcct atcggttcaa cggcatcggc gtgacccaga atgtgctgta cgagaaccag 2760

aagctgatcg ccaatcagtt taactccgcc atcggcaaga tccaggactc tctgagctcc 2820

acagcaagcg ccctgggcaa gctgcaggat gtggtgaatc agaacgccca ggccctgaat 2880

accctggtga agcagctgtc tagcaacttc ggcgccatct cctctgtgct gaatgatatc 2940

ctgagcaggc tggacaaggt ggaggcagag gtgcagatcg accggctgat cacaggcaga 3000

ctgcagtccc tgcagaccta cgtgacacag cagctgatca gggcagcaga gatcagggca 3060

tctgccaatc tggccgccac caagatgagc gagtgcgtgc tgggccagtc caagagagtg 3120

gacttttgtg gcaagggcta tcacctgatg agcttcccac agtccgcccc tcacggagtg 3180

gtgtttctgc acgtgaccta cgtgccagcc caggagaaga acttcaccac agcaccagca 3240

atctgccacg atggcaaggc acactttcct agggagggcg tgttcgtgag caacggcacc 3300

cactggtttg tgacacagcg caatttctac gagccacaga tcatcaccac agacaataca 3360

ttcgtgtccg gcaactgtga cgtggtcatc ggcatcgtga acaataccgt gtatgatcct 3420

ctgcagccag agctggactc ttttaaggag gagctggata agtacttcaa gaatcacacc 3480

agccccgacg tggatctggg cgacatctct ggcatcaatg ccagcgtggt gaacatccag 3540

aaggagatcg acaggctgaa cgaggtggcc aagaatctga acgagtccct gatcgatctg 3600

caggagctgg gcaagtatga gcagtacatc aagtggccct ggtatatctg gctgggcttc 3660

atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgtat gacaagctgc 3720

tgttcctgcc tgaagggctg ctgttcttgt ggcagctgct gtaagtttga tgaggacgat 3780

agcgagcctg tgctgaaggg cgtgaagctg cactacacca ccggtctgca gctagctcga 3840

gtc 3843

<210> 12

<211> 1281

<212> PRT

<213> novel coronavirus (SARS-CoV-2 virus)

<400> 12

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

675 680 685

Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

705 710 715 720

Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

965 970 975

Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln

980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn

1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys

1025 1030 1035

Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val

1055 1060 1065

Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His

1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn

1085 1090 1095

Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln

1100 1105 1110

Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val

1115 1120 1125

Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro

1130 1135 1140

Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1145 1150 1155

His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn

1160 1165 1170

Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu

1175 1180 1185

Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu

1190 1195 1200

Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu

1205 1210 1215

Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met

1220 1225 1230

Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys

1235 1240 1245

Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro

1250 1255 1260

Val Leu Lys Gly Val Lys Leu His Tyr Thr Thr Gly Leu Gln Leu

1265 1270 1275

Ala Arg Val

1280

<210> 13

<211> 2415

<212> DNA

<213> Artificial sequence

<400> 13

atgtcaagct cttcctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60

attgaggaac aggccaagac atttttggac aagtttaacc acgaagccga agacctgttc 120

tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180

aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacacttgcc 240

caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300

cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360

aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420

tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480

aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540

gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600

gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660

cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720

catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780

tgcctccctg ctcatttgct tggtgatatg tggggtagat tttggacaaa tctgtactct 840

ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900

gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960

cctaatatga ctcaaggatt ctgggaaaat tccatgctaa cggacccagg aaatgttcag 1020

aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080

tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140

tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200

catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260

attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320

aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380

tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440

aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500

gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560

taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620

aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680

ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740

gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800

aattcttttg tgggatggag taccgactgg agtccatatg cagaccaaag catcaaagtg 1860

aggataagcc taaaatcagc tcttggagat aaagcatatg aatggaacga caatgaaatg 1920

tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980

cagatgattc tttttgggga ggaggatgtg cgagtggcta atttgaaacc aagaatctcc 2040

tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100

gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gaatgacaac 2160

agcctagagt ttctggggat acagccaaca cttggacctc ctaaccagcc ccctgtttcc 2220

atatggctga ttgtttttgg agttgtgatg ggagtgatag tggttggcat tgtcatcctg 2280

atcttcactg ggatcagaga tcggaagaag aaaaataaag caagaagtgg agaaaatcct 2340

tatgcctcca tcgatattag caaaggagaa aataatccag gattccaaaa cactgatgat 2400

gttcagacct ccttt 2415

<210> 14

<211> 805

<212> PRT

<213> Artificial sequence

<400> 14

Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala

1 5 10 15

Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe

20 25 30

Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp

35 40 45

Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn

50 55 60

Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala

65 70 75 80

Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln

85 90 95

Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys

100 105 110

Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser

115 120 125

Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu

130 135 140

Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu

145 150 155 160

Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu

165 170 175

Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg

180 185 190

Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu

195 200 205

Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu

210 215 220

Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu

225 230 235 240

His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile

245 250 255

Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly

260 265 270

Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys

275 280 285

Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala

290 295 300

Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu

305 310 315 320

Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro

325 330 335

Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly

340 345 350

Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp

355 360 365

Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala

370 375 380

Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe

385 390 395 400

His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys

405 410 415

His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn

420 425 430

Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly

435 440 445

Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe

450 455 460

Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met

465 470 475 480

Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr

485 490 495

Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe

500 505 510

Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala

515 520 525

Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile

530 535 540

Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu

545 550 555 560

Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala

565 570 575

Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe

580 585 590

Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr

595 600 605

Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu

610 615 620

Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met

625 630 635 640

Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu

645 650 655

Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val

660 665 670

Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro

675 680 685

Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile

690 695 700

Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn

705 710 715 720

Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln

725 730 735

Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val

740 745 750

Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg

755 760 765

Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile

770 775 780

Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr Asp Asp

785 790 795 800

Val Gln Thr Ser Phe

805

Claims (10)

1. An immunogenic peptide against the novel coronavirus SARS-CoV-2, comprising the RBD region of the spike protein S of SARS-CoV-2 virus, wherein the RBD region is further modified with cysteine to form the sRBD region.

2. The immunogenic peptide of claim 1, wherein the amino acid sequence of the RBD region is set forth in SEQ ID NO. 2 and the amino acid sequence of the cysteine-modified sRBD region is set forth in SEQ ID NO. 4.

3. The immunogenic peptide of claim 1, wherein the RBD region or cysteine-modified sRBD region is comprised in a fusion peptide, e.g. the portion fused thereto comprises: the fusion peptide is a protein derived from virus or host, transferrin (Fn), HIV p24, and a stem part of a enveloped virus, such as influenza HA2, gp41 of AIDS virus, an antibody Fc segment, GM-CSF, IL-21, CD40L or CD40 antibody, and the sequence of the fusion peptide is shown as SEQ ID NO:8 or SEQ ID NO: 10.

4. A nucleotide molecule encoding the immunogenic peptide of any one of claims 1-3.

5. The nucleotide molecule of claim 4, wherein the coding sequence of the RBD region is shown as SEQ ID NO. 1, the coding sequence of the cysteine modified RBD region is shown as SEQ ID NO. 3, or the sequence of the nucleotide molecule is shown as SEQ ID NO. 7 or SEQ ID NO. 9.

6. A vector comprising the nucleotide molecule of any one of claims 4-5.

7. A host cell comprising the vector of claim 6 and capable of expressing the immunogenic peptide of any one of claims 1-3.

8. A vaccine against the novel coronavirus SARS-CoV-2, comprising the immunogenic peptide of any one of claims 1-3, the nucleotide molecule of any one of claims 4-5, the vector of claim 6, and/or the host cell of claim 7.

9. Use of the immunogenic peptide according to any one of claims 1 to 3, the nucleotide molecule according to any one of claims 4 to 5, the vector according to claim 6 and/or the host cell according to claim 7 for the preparation of a vaccine for the prevention or treatment of the novel coronavirus SARS-CoV-2.

10. A method of making a vaccine against the novel coronavirus SARS-CoV-2, the method comprising:

(a) providing a host cell comprising the immunogenic peptide of any one of claims 1-3, the nucleotide molecule of any one of claims 4-5, the vector of claim 6, and/or the host cell of claim 7;

(b) combining the active substance provided in (a) with an immunologically or pharmaceutically acceptable carrier.

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